A Triple Line of Defense: The Role of the Group 1 RFX Transcription Factors in the Inner Ear

项目摘要 为了成功地开发再生疗法来治疗大部分受影响的个体， 听力和前庭疾病，我们必须首先了解必要的转录调控网络， 内耳毛细胞的发育和维持。因此，国家耳聋和其他疾病研究所（National Institute on Deafness and Other） 沟通障碍（NIDCD）已将其第一优先事项（2017-2021年战略计划的优先领域1）确定为 识别参与外周神经发育的分子以及遗传和表观遗传变化 以及中枢听觉和前庭通路。第1组调节因子X（RFX）转录的成员 TF家族（RFX 1、RFX 2和RFX 3）被认为是纤毛发生的主要调节因子， 我们的实验室最近发现，它在毛细胞发育中起着关键作用。具体地，Rfx 1的表达 而Rfx 3（Rfx 1/3）是外毛细胞终末分化和发病后存活所必需的 听觉的。没有Rfx 1/3表达的小鼠具有晚期、轻度前庭表型。我们已经发现，Rfx 2， 组-1 RFX TF的最后成员，也在听觉和前庭毛细胞中表达。所以我们 假设在Rfx 1/3条件性敲除（cKO）小鼠中Rfx 2补偿Rfx 1/3的损失， 为了揭示组-1 RFXTF在毛细胞发育中的全部作用，所有三种组-1 RFXTF必须 的页面不存在或我们预测毛细胞中Rfx 1、Rfx 2和Rfx 3（Rfx 1/2/3）表达的缺失将导致严重的 平面细胞极性（PCP）缺陷以及强大的听觉和前庭功能障碍。具体目标1将 通过详细的组织学研究阐明组1 RFX TF在早期毛细胞发育和PCP中的作用 Rfx 2Gt（基因陷阱敲除模型）、Rfx 1/2/3 cKO和对照同窝小鼠的分析。此外，本发明还 我们将通过X-gal染色内耳完成Rfx 2表达的时空表征 路段具体目标2将通过进行以下研究来探索第1组RFX TF在前庭功能中的作用： 对Rfx 2Gt、Rfx 1/2/3 cKO和对照同窝小鼠进行行为和电生理测试。具体 目的3将确定前庭毛细胞发育中第1组RFX TF的下游靶点， 单细胞RNA测序和RNAScope直接靶点验证。拟议的工作具有影响力 因为它将确定RFX TF是否也会调节内耳毛细胞中的纤毛发生和PCP， 对内耳功能重要的基因，并有助于导致TF发展的工作主体 用于HC再生的鸡尾酒。

PROJECT SUMMARY In order to successfully develop regenerative therapies to treat a large percentage of individuals affected by hearing and vestibular disorders, we must first understand the transcriptional regulatory networks necessary for inner ear hair cell development and maintenance. Thus, the National Institute on Deafness and Other Communication Disorders (NIDCD) has set its first priority (Priority Area 1 of the 2017-2021 Strategic Plan) to identify the molecules and genetic and epigenetic changes that take part in the development of the peripheral and central auditory and vestibular pathways. Members of the group-1 Regulatory Factor X (RFX) transcription factor (TF) family (RFX1, RFX,2 and RFX3), which are known as master regulators of ciliogenesis, have been recently found by our laboratory to play a key role in hair cell development. Specifically, the expression of Rfx1 and Rfx3 (Rfx1/3) is necessary for the terminal differentiation of outer hair cells and their survival after the onset of hearing. Mice without Rfx1/3 expression have a late, mild vestibular phenotype. We have found that Rfx2, the final member of the group-1 RFX TFs, is also expressed in the auditory and vestibular hair cells. Therefore, we hypothesize that Rfx2 compensates for the loss of Rfx1/3 in the Rfx1/3 conditional knockout (cKO) mice and that to uncover the full role of the group-1 RFX TFs in hair cell development, all three group-1 RFX TFs must be deleted. We predict that loss of expression of Rfx1, Rfx2, and Rfx3 (Rfx1/2/3) from hair cells will result in a severe planar cell polarity (PCP) defect as well as robust auditory and vestibular dysfunction. Specific Aim 1 will elucidate the role of the group-1 RFX TFs in early hair cell development and PCP through detailed histological analyses of the Rfx2Gt (a gene trap knockout model), Rfx1/2/3 cKO, and control littermate mice. Additionally, we will complete the spatiotemporal characterization of the expression of Rfx2 by X-gal staining of inner ear sections. Specific Aim 2 will explore the role the group-1 RFX TFs in vestibular function by conducting behavioral and electrophysiological testing on the Rfx2Gt, Rfx1/2/3 cKO, and control littermate mice. Specific Aim 3 will identify downstream targets of the group-1 RFX TFs in vestibular hair cell development with the use of single cell RNA-sequencing and validation of direct targets with RNAScope. The proposed work is impactful as it will determine whether RFX TFs will also regulate ciliogenesis and PCP in the inner ear HCs, identify new genes important for inner ear function, and contribute to the body of work leading to the development of TF cocktails for HC regeneration.